G protein-coupled receptor kinases in hypertension: physiology, pathogenesis, and therapeutic targets

G protein-coupled receptors (GPCRs) mediate cellular responses to a myriad of hormones and neurotransmitters that play vital roles in the regulation of physiological processes such as blood pressure. In organs such as the artery and kidney, hormones or neurotransmitters, such as angiotensin II (Ang II), dopamine, epinephrine, and norepinephrine exert their functions via their receptors, with the ultimate effect of keeping normal vascular reactivity, normal body sodium, and normal blood pressure. GPCR kinases (GRKs) exert their biological functions, by mediating the regulation of agonist-occupied GPCRs, non-GPCRs, or non-receptor substrates. In particular, increasing number of studies show that aberrant expression and activity of GRKs in the cardiovascular system and kidney inhibit or stimulate GPCRs (e.g., dopamine receptors, Ang II receptors, and α- and β-adrenergic receptors), resulting in hypertension. Current studies focus on the effect of selective GRK inhibitors in cardiovascular diseases, including hypertension. Moreover, genetic studies show that GRK gene variants are associated with essential hypertension, blood pressure response to antihypertensive medicines, and adverse cardiovascular outcomes of antihypertensive treatment. In this review, we present a comprehensive overview of GRK-mediated regulation of blood pressure, role of GRKs in the pathogenesis of hypertension, and highlight potential strategies for the treatment of hypertension. Schematic representation of GPCR desensitization process. Activation of GPCRs begins with the binding of an agonist to its corresponding receptor. Then G proteins activate downstream effectors that are mediated by various signaling pathways. GPCR signaling is halted by GRK-mediated receptor phosphorylation, which causes receptor internalization through β-arrestin.

and the number of deaths attributable to high blood pressure increased 90.1% from 2010 to 2020 [3].Hypertension is a major public health problem and challenges worldwide [4].
Cells communicate with each other by receiving and sending through a myriad of extracellular proteins/molecules, such as neurotransmitters, chemoattractants, and hormones, which bind to receptors and drive the initiation of multiple intracellular signaling routes.G protein-coupled receptors (GPCRs) are the largest superfamily of cell-surface receptors and the most diverse group of proteins involved in transmembrane signal transduction [5,6].They share conserved seven α-helical structured transmembrane domains, extracellular ligand-binding domains, and carboxy-terminal intracellular domains.GPCRs, including adrenergic receptors, angiotensin II (Ang II) receptors, and dopamine receptors, control a variety of physiological processes involved in the regulation of blood pressure [7,8].Cell dysfunction, induced by aberrant GPCRs, including abnormal phosphorylation, trafficking, and expression, causes hypertension by various mechanisms [8][9][10].
The phosphorylation and desensitization of agonistoccupied GPCRs are primarily mediated by GPCR kinases (GRKs) [11].As their name implies, GRKs are defined by their actions on GPCR phosphorylation, receptor internalization, and subsequent signal termination (Fig. 1).Abnormalities of GRKs are associated with cardiovascular diseases, including hypertension [12,13].A number of studies have shown that GRKs play a vital role in the regulation of blood pressure; aberrant expression and/or activity of GRKs cause hypertension and are associated with impaired response to antihypertensive treatment [14][15][16][17].There are abundant epidemiological and animal studies reinforcing the role of GRKs and their underlying mechanisms in the regulation of blood pressure.Therefore, in this article, we review our evolving understanding of GRK-mediated regulation of blood pressure and highlight potential strategies for targeting GRKs in the prevention and treatment of hypertension.
The Grk family GRK-mediated receptor phosphorylation is one of the wellcharacterized mechanisms for GPCR desensitization.There Graphical Abstract Schematic representation of GPCR desensitization process.Activation of GPCRs begins with the binding of an agonist to its corresponding receptor.Then G proteins activate downstream effectors that are mediated by various signaling pathways.GPCR signaling is halted by GRK-mediated receptor phosphorylation, which causes receptor internalization through βarrestin.
are more than 800 distinct GPCRs in the human genome but of the vital regulators of agonist-induced phosphorylation of GPCRs, only seven GRKs (GRK1-7) have been identified [18].Based on their sequence similarity and gene structure, the serine/threonine kinase GRK family encompasses seven protein isoforms that are classified into three subfamilies.GRK1 and GRK7 belong to the GRK1-like subfamily, also known as visual or rhodopsin-specific GRKs.GRK2 (β-adrenergic receptor kinase 1, β-ARK1) and GRK3 (β-ARK2) belong to the GRK2-like subfamily, also known as β-adrenergic receptor (β-AR) kinase subfamily.GRK4, GRK5, and GRK6 belong to the GRK4-like subfamily [17,18].
The seven GRKs share a common general protein structure with a central catalytic domain (~270 amino acids), flanked by an amino-terminal (N-terminal) domain (~185 amino acids) and a carboxyl terminal (C-terminal) domain (~105 to 230 amino acids) [19].The N-terminus, a well-conserved domain between GRK members, is vital for the selective recognition of the activated receptor and also contains a region termed as a regulator of G protein signaling homology (RH) domain (~120 amino acids).However, the C-terminus is divergent among GRK subfamilies and contributes to their subcellular membrane localization and agonist-dependent translocation [19,20].
In addition to GPCRs, GRKs also regulate the phosphorylation or expression of other non-GPCR receptors, such as adiponectin receptor 1 (AdipoR1) and insulin-like growth factor-1 receptor [21,22].Moreover, GRKs regulate nonreceptor substrates, including signaling proteins, such as insulin receptor substrate-1, nuclear proteins, such as histone deacetylase 5, and transcription factors, such as IκBα, among others [23][24][25][26].We also reported that GRK4 increases the phosphorylation of signal transducer and activator of transcription 1 and HDAC4 [27,28].It should also be noted that kinase activity is not necessary for some GRK-mediated functions.For example, GRK4 regulates cilia and kidney development independent of its kinase function [29].
All GRKs are primarily localized in the cytosol and plasma membrane.However, the tissue distribution of the seven GRK subtypes is different from each other.GRK2, GRK3, GRK5, and GRK6 are ubiquitously distributed among mammalian tissues [17].GRK1, GRK4, and GRK7 are expressed in specific tissues.GRK1 and GRK7 are expressed exclusively in the retina, whereas GRK4 is expressed in a few organs [17,30].These indicate that GRKs may exert different physiological functions based on their tissue distribution.

Role of Grks on blood pressure regulation
Many studies have shown that GRKs exert their biological functions by mediating the regulation of agonist-occupied GPCRs, non-GPCR receptors, and non-receptor substrates (Table 1).Several organs, including the artery, heart, and kidney, are involved in GRK-mediated blood pressure regulation (Fig. 2).The expression and/or activity of GRKs are aberrant in the hypertensive humans and animal models of hypertension.Because GRK1 and GRK7 are expressed only in the retina, only the role of the other five GRKs in the regulation of blood pressure have been studied.

GRK2
Role of GRK2 in the regulation of blood pressure GRK2 is ubiquitously expressed in mammalian organs and tissues, including the artery, heart, and kidney.GRK2 is not only a key regulator of vascular responsivity in resistance arteries but also regulates myocardial contractility [31,32], and renal function (vide infra).
GRK2 regulates GPCR-mediated vascular signaling, which participates in the regulation of blood pressure.Global knockdown of GRK2 [33] or vascular smooth muscle (VSM)-targeted overexpression of GRK2 in mice increases resting blood pressure [34].This apparent inconsistent effect on blood pressure may be related to the fact that the alteration in GRK2 expression (increased or decreased) can lead to the imbalance of vasoconstrictor and vasodilator effects, resulting in hypertension [33].GRK2 negatively regulates GPCRs such as endothelin type A receptor [35], P2Y2 receptor [36,37], angiotensin II type 1 receptor (AT 1 R) [38] and α 1D -adrenergic receptor [39], which should result in vasodilation.By contrast, GRK2 also negatively regulates β 2 -adrenergic receptor [40] and dopamine D 1 receptor (D 1 R)/adenylyl cyclase signaling [41], which should result in vasoconstriction.The rebound hypertension in mice that occurs after withdrawal of clonidine, an α 1D, α 2A-C adrenergic receptor agonist, is mitigated by an increase in nitric oxide bioavailability, an effect that is prevented by suppression of GRK2 activity [42].However, one group reported that global GRK2 knockdown impaired kidney development, and increased blood pressure [43] while another group reported that Grk2 +/− mice have normal blood pressure and attenuated response to the hypertensive effect of Ang II infusion [31].Therefore, the overall effect of GRK2 on blood pressure is difficult to predict (vide infra).
GRK2 directly interacts with the epithelial sodium channel (ENaC) [44], which increases the reabsorption of filtered sodium across the luminal membrane of the principal cells of the aldosterone-sensitive distal nephron [45].GRK2 positively regulates ENaC activity by phosphorylating Nedd4 and Nedd4-2, two ubiquitin-protein ligases binding with the channels, to prevent their ability to inhibit epithelial channel activity [46].The dopaminergic system plays a vital role in the regulation of sodium excretion.The activation of GRK2 by some factors such as oxidative stress and insulin impairs the ability of renal D 1 R to inhibit sodium transport [47,48] that leads to the development of hypertension [8].We have also reported that exposure to maternal diabetes mellitus causes renal D 1 R dysfunction and hypertension in adult rat offspring by increasing GRK2 activity [49].Thus, GRK2 can increase blood pressure by increasing renal sodium transport.
GRK2 also regulates receptor-mediated signaling in the heart.GRK2 phosphorylates cardiac β 2 -adrenergic receptor and increases G i -biased signaling, which contributes to heart failure [50].However, GRK2 inhibition increases βadrenergic receptor-dependent cardiomyocyte and cardiac contractility and reverses cardiac dysfunction in heart failure models [51].GRK2 also regulates the desensitization of other cardiac receptors, such as AdipoR1 and κ-opioid receptor [52,53].In addition, GRK2 participates in the regulation of other physiological or pathological functions in the heart, including fatty acid metabolism, production of NLRP3 inflammasome and oxidative stress, and modulation of insulin signaling [54][55][56].How, these GRK2-mediated effects in the heart regulate blood pressure need to be studied further.

HEK293 cells
Grk5 overexpression increases the angiotensin II-induced AT 1 R phosphorylation [38] β-AR VSM [122]; heart [32] VSM-Grk5 over-expression increases blood pressure to a greater extent in male than female mice; β-AR stimulation induced vasoconstriction in male mice but decreased β-ARinduced vasodilation and increased angiotensin II sensitivity in female mice [122]; Cardiac-specific Grk5 expression increases β-AR desensitization but does not affect the contractile response to angiotensin II [32] Intestinal epithelial cells GRK6 antibodies prevents the desensitization-mediated loss of D 1 R inhibition of Cl/ HCO3 -exchanger activity [130] Non-GPCR receptor substrates GRK4 adipoR1 Renal cortex; rat RPT cells hGRK4γ 142V causes adipoR1 phosphorylation and desensitization [21] Grk2 or VSM-specific ablation of Grk2 protein expression in mice has no effect on the increased blood pressure in the twokidney, one-clip (2K1C) model, which may be due to a balance between the increased β-AR-mediated dilation and the enhancement of α 1D -AR-stimulated vasoconstriction [39].
The reason for the inconsistent reports on the effect of Grk2 knockdown on blood pressure remains unclear.We speculate some possibilities: 1) Different methods of Grk2 knockdown may produce different effects.Global knockdown of GRK2 using a small hairpin (sh) RNA leads to hypertension [33,43], a partial deficiency of GRK2 protects against Ang II-induced hypertension [31]; VSM-specific Grk2 knockdown has no effect on blood pressure [39].2) GRK2 regulates both vasoconstriction and dilation.For example, GRK2 deficiency increases phenylephrine, but not KCl-mediated constriction and isoproterenolmediated vasodilation [33].The dominant effect induced by GRK2 knockdown may determine the physiological or pathological phenotype.3) Duration of Grk2 silencing.There is a balance between vasoconstriction and vasodilation in Grk2 knockdown mice.Twelve to 14 weeks of Grk2 knockdown using small hairpin RNA causes a shift toward vasoconstriction and subsequent hypertension [33].Additionally, activated renal AT 1 R-mediated signaling also plays an important role in the development of hypertension after six months of Grk2 knockdown [43].However, a longer (nine months) duration of Grk2 knockdown was reported to lead to a nitric oxide-dependent vasodilation which overcomes vasoconstriction, making Grk2 +/ − mice resistant to the development of hypertension [31].4) It is possible that the different effects on blood pressure induced by Grk2 knockdown may be also attributed to experimental conditions such as animal species and shRNA induction.
The depletion of Grk2 expression in aortic smooth muscle cells also impairs the proliferative effect of endothelin and Ang II [57], that should prevent the increase in blood pressure.Grk2 knockdown in the endothelial cells of mesenteric arterioles promotes insulin-induced vasodilation and insulin-stimulated signaling pathway in SHRs; this effect is prevented by upregulation of Grk2 [58].Selective deletion of Grk2 in vascular endothelium of mice also blunts the vasoconstriction induced by the α 1 -adrenergic receptor agonist phenylephrine and other vasoconstrictors (serotonin, oxytocin, and KCl) [59].
By contrast, transgenic mice with VSM-targeted Grk2 over-expression have higher resting blood pressure than their wild-type littermates [34].This is consistent with another study, which found that VSM Grk2 over-expression leads to increased systolic blood pressure (SBP).Moreover, epinephrine injection increased SBP to a greater extent in the VSM-Grk2 over-expressing mice than wild-type control mice [60].However, cardiac-specific Grk2 expression attenuates Ang II-induced increase in contractility and blood pressure [32], consistent with the pro-and antihypertensive effects of GRK2 that are tissue-dependent.

GRK2 expression is increased in animal models of hypertension
The expression of Grk2 mRNA is higher in the aorta but lower in the kidney of spontaneously hypertensive rats (SHRs) than Wistar-Kyoto (WKY) rats [61,62].GRK2 protein expressions in lymphocytes and VSMs are also higher in SHRs than Wistar-and WKY rats; vascular GRK2 protein expression is also higher in hypertensive Dahl saltsensitive than normotensive Dahl salt-resistant rats [63].The expressions of GRK2 and adrenergic receptors are decreased in the aorta but not in mesenteric arteries of NGnitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats, that is accompanied by decreased α 1 -adrenergic receptor-induced vasoconstriction along with increased β 2adrenergic receptor-mediated vasodilation; these changes are the opposite to those found in the aortas of SHRs [64].In addition, renal GRK2 expression is increased in L-NAME-induced hypertensive rats.However, renal but not left ventricular GRK2 expression is decreased in SHRs [62].

GRK2 and human hypertension
Studies have shown abnormal GRK2 expression and/or activity in the hypertensive state.GRK activity and GRK2 expression in lymphocytes are increased in hypertensive relative to normotensive humans [14].The increase in GRK2 protein expression in lymphocytes from hypertensive subjects is positively associated with blood pressure and inversely associated with β-adrenergic agonist-mediated increase in adenylyl cyclase activity [14,65].Izzo et al. also reported that GRK2 expression is increased in blood lymphocytes of hypertensive patients and the impaired β-ARmediated vasorelaxation in the brachial artery is restored by treatment with heparin, a nonspecific GRK inhibitor [66].Further studies showed that in Black Americans, GRK2 but not GRK5 protein expression is increased 2-fold and GRK activity is increased >40% in the lymphocytes, which correlated with increasing blood pressure [67].A case-control study in Han Chinese showed that the dominant model (CC vs. CT + TT) of rs1894111 polymorphism in the ADRBK1 (GRK2) gene is associated with low-renin hypertension [68].In addition, abnormal GRK2 expression or activity is associated with other types of hypertension.For example, elevated GRK2 levels in the umbilical vasculature are associated with elevated blood pressure in subjects with gestational hypertension and preeclampsia [69], but this may be a compensatory rather than a causative effect.Insufficient GRK2 activity compromises spiral artery remodeling and initiates necrotic events in the placenta, thereby causing preeclampsia [70].

GRK3
GRK3 is ubiquitously expressed in mammalian organs and tissues.There are limited studies on the association between GRK3 and hypertension.GRK3 expression in cardiac myocytes progressively increases in 6-, 12-, and 24-monthold spontaneously hypertensive heart failure rats and is significantly higher than in control rats [71].Grk3 mRNA levels in human lymphocytes are inversely associated with

Regulation of blood pressure by GRKs
Kidney: GRK2-6

Artery: GRK2-6
Heart: GRK2-6 Fig. SBP and diastolic blood pressure (DBP) [72], suggesting that GRK3 may play a beneficial role in the regulation of blood pressure.This is, at least in part, supported by findings in transgenic mice.Cardiac myocyte-restricted expression of the carboxyl-terminal fragment of Grk3, a competitive inhibitor of Grk3, results in a phenotype with elevated SBP and DBP but without alterations of heart rate in conscious, unrestrained mice [73].Further studies found that the cardiomyocyte-restricted inhibition of Grk3 in mice does not alter the hypertrophic response but attenuates cardiac dysfunction and heart failure with chronic pressure overload [74].It should be noted that some GPCRs that regulate blood pressure, such as dopamine receptors and β-ARs are also regulated by GRK3 [75,76].Grk3 expressed in HEK293 cells augments the agonist-induced AT 1 R or D 1 R phosphorylation, causing their desensitization [41].However, the physiological consequences of altered GRK3 expression is unclear.Renal GRK3 expression is not different between WKY rats and SHRs but renal GRK3 mRNA is increased in the kidneys of rats made hypertensive with L-NAME [62].The role of GRK3 in the regulation of blood pressure needs to be studied further.

GRK4
Effect of GRK4 on blood pressure GRK4 is expressed to a greater extent in the testes and myometrium and to a lesser extent in other tissues such as the brain and kidney [13].We also reported its expression in the artery and heart [28,77], and found that renal and cardiac GRK4 levels are elevated by kidney ischemiareperfusion injury and myocardial infarction, respectively [27,28].
The role of GRK4 in the regulation of blood pressure is confirmed in several animal models of hypertension.Global Grk4 knockout mice have lower SBP and DBP than Grk4 wild-type (WT) mice [78].Selective renal Grk4 knockdown, via chronic renal cortical interstitial-selective infusion of Grk4 antisense oligodeoxynucleotides, increases urine flow and ameliorates the sodium retention and elevated blood pressure of SHRs [79,80].We have reported that down-regulation of renal GRK4 expression, via ultrasound-targeted microbubble destruction-delivered Grk4 small interfering RNA (siRNA), decreases blood pressure, accompanied with increased sodium excretion in SHRs [21,81].
In contrast to the lowering of blood pressure caused by the silencing of Grk4 [78][79][80][81][82], the over-expression of human Grk4 gene variants (hGrk4γ 65L, hGrk4γ 142V, or hGrk4γ 486V) in mice has the opposite effect on blood pressure.On normal salt diet, transgenic mice with over-expression of human GRK4γ (hGRK4γ) variant 142V have higher blood pressure than WT transgenic mice [15,77,78,82].However, hGrk4γ 486V [83] and hGrk4γ 65L transgenic mice have normal blood pressure on a normal-salt diet but have elevated blood pressure on a highsalt diet, indicating that hGRK4γ 486V and hGRK4γ 65L transgenic mice have salt-sensitive hypertension while hGrk4γ 142V transgenic mice have salt-resistant hypertension.
The reason why different GRK4 variants exhibit different phenotypes in blood pressure regulation is still unknown.Our previous study found that the salt sensitivity of hGRK4γ 486V mice is due, in part, to increased renal reactive oxygen species production, which can be attributed to defective renal antioxidant mechanisms [83].However, the main reason for the different phenotypes of different GRK4 variants could be due to the different structures of the GRK4 variants.We presume that the polymorphisms alter the structure of GRK4, that modulate its kinase activity, and affect substrate-regulated biological responses that subsequently cause hypertension.Allen et al. reported the overall crystal structure of GRK4α A486V and differences in the structure of GRK4α A486V from other GRKs.They found that relative to WT GRK4α, GRK4α A486V has an increased rate of autophosphorylation of a number of residues, which is due to a defective lag in the absence of preincubation with ATP [84].However, the mechanism by which different GRK4 polymorphisms affect enzymatic activity needs more investigation in future studies.
The control of blood pressure by GRK4 is due in part to its regulation of renal dopamine receptors.In human renal proximal tubule cells, GRK4 constitutively phosphorylates the D 1 R, even in the absence of agonist activation, causing its internalization and constitutive desensitization [85].Treatment with heparin, an inhibitor of GRK activity, or antisense oligonucleotides (GRK4 > GRK2) blunt the agonist-induced D 1 R desensitization [86].In human essential hypertension, single nucleotide polymorphisms (SNPs) in GRK4γ increase GRK activity and D 1 R serine phosphorylation, causing the uncoupling of D 1 R from its G protein/effector enzyme complex in the renal proximal tubule [15].GRK4-mediated regulation of D 1 R expression or activity has been confirmed in several animal models of hypertension.We have reported that GKR4 impairs renal tubular and arterial D 1 R expression and D 1 R-mediated sodium excretion and vasodilation in offspring with hypertension related to in utero exposure to some factors such as fine particulate matter, e.g., PM 2.5 , lipopolysaccharide, and cold stress [87][88][89].The impairment of the D 1 R function as part of the insulin resistance in type 2 diabetes mellitus in mice has also been related to increased GRK4 expression [90].GRK4 also regulates the phosphorylation and function of the dopamine D 3 receptor (D 3 R) in human proximal tubule cells [91].However, the role of GRK4 in D 3 R-mediated regulation of blood pressure remains unclear.
GRK4 can also control blood pressure by interacting with the AT 1 R. hGrk4γ 142V phosphorylates HDAC1 and promotes its export from the nucleus to the cytoplasm resulting in an increase in the transcription of AT 1 R, presumably by decreasing its deacetylation [78].AT 1 R blockade or the deletion of the Agtr1a gene normalizes hypertension in Grk4γ 142V mice [78].We have reported that GRK4 is expressed in VSMCs of the aorta.AT 1 R expression is higher in the aorta of Grk4γ 142V transgenic mice than WT mice.Ang II-mediated vasoconstriction of the aorta is greater in Grk4γ 142V mice than in WT mice [77].GRK4-positive regulation of AT 1 R expression and function is also found in other hypertensive animal models, including the male hypertensive offspring of male Sprague-Dawley rats that were exposed to fine particulate matter with an aerodynamic diameter of ≤2.5 μm [92].We have also recently reported that GRK4 increases the phosphorylation of angiotensin II type 2 receptor (AT 2 R) which impairs renal AT 2 R-mediated diuresis and natriuresis, which participate in the GRK4-mediated regulation of blood pressure [93].In addition to dopamine and Ang II receptors, GRK4 also regulates other receptors such as the endothelin type B receptor (ETBR) and AdipoR1.Compared with WT mice, hGRK4γ 142V mice have increased phosphorylation of renal ETBR and AdipoR1 and impaired ETBR-and AdipoR1-induced diuresis and natriuresis [21,82].By contrast, renal-selective GRK4 knockdown normalizes the increased ETBR and AdipoR1 phosphorylation, and partially or fully restores the ETBR-and AdipoR1-mediated increase in sodium excretion and reduction of blood pressure in SHRs [21,82].

GRK4 in hypertensive animal models
Basal GRK4 expression and activity in the kidney and artery are higher in SHRs than in WKY rats [61,79,80,82,93].The increased GRK4 expression in the renal and artery in SHRs is organ-specific because there is no difference in cardiac GRK4 expression between WKY and SHRs [79].Similar results are found in kidneys of humans with essential hypertension.Moreover, the increased activity of GRK4 in the kidneys of hypertensive subjects is caused not by increased renal GRK4 protein expression but rather by constitutively active variants of GRK4 [15].The role of GRK4 in hypertension is further confirmed by studies involving renal Grk4 depletion, which efficiently reduces GRK4 expression and lowers blood pressure in SHRs, accompanied with increased urine volume and sodium excretion [21,[79][80][81][82].In addition, GRK4 expression and activity in the kidney and artery are increased in other animal models of hypertension [87][88][89][94][95][96].
GRK4 variants are also associated with salt sensitivity.In a Japanese cohort, the genotype and allele frequencies of GRK4 SNPs, including R65L, A142V, and A486V, are higher in salt-sensitive than in salt-resistant hypertensive subjects.Sodium excretion was reported to be inversely related to the number of GRK4 variants in hypertensive persons, and the natriuretic response to dopaminergic stimulation is impaired in normotensive persons having three or more GRK4 gene variants [99].A large genome-wide association study in 15,034 Korean adults investigated the correlation of 1282 candidate SNPs with dietary sodium intake and found that the GRK4 R65L TT genotype is inversely associated with salt-sensitive hypertension risk [103].Moreover, GRK4 polymorphisms are also associated with salt sensitivity in normotensive subjects [107,116].The GRK4 65L allele is associated with impaired renal sodium handling in response to stress in Black normotensive adolescents [117].

GRK5
GRK5 is ubiquitously expressed in mammalian organs and tissues, including the heart, lung, and retina [118].GRK5 is involved in cardiovascular diseases, including cardiac fibrosis, heart failure, and hypertension [12,[119][120][121][122][123][124].GRK5 mRNA and protein levels are increased in the aorta of Ang II-and norepinephrine-induced hypertensive rats [121].Gkr5 mRNA expression is also increased in the left ventricle and kidney of rats with hypertension induced by L-NAME [62].However, in this report, the mRNA levels of

Single-locus
Correlates with blood pressure response to metoprolol among hypertensive male subjects [111] Black subjects Single-locus Predicts blood pressure response to dietary modification in Black subjects with mild-to-moderate hypertension [169] American

Single-locus
Correlates with increased risk for adverse cardiovascular outcomes [164] Japanese Single-locus Correlates with hypertension; Carriers of hGRK4 142V have a greater decrease in SBP in response to ARBs [165] Chinese Single-locus Hypertensive subjects with GRK4 A142V are more likely to be non-dippers [166] A486V (rs1801058) Italian Single-locus Associates with mild hypertension [102] Euro-Australian African American

Single-locus
Negatively correlates with hypertension in nonobese subjects [112] European Single-locus Correlates with salt sensitivity of blood pressure [107] African Brazilian Two-locus NOS3-GRK4 interaction is related to DBP levels [98] American

Single-locus
Correlates with increased risk for adverse cardiovascular outcomes [164] Japanese Single-locus Correlates with hypertension; Carriers of hGRK4 486V were less to achieve the blood pressure goal in response to an ARB [165] Korean Single-locus A high sodium intake increases the obesity risk in children with GRK4 A486V regardless of sex [116] Korean Multi-locus A high sodium intake increases the obesity risk in boys with the combination of GRK4 A486V, ACE, and SLC12A3 variants [116] Korean Two-locus The combination of GRK4 A486V and CYP11B2 variants increases the obesity risk in girls when sodium intake is increased [116] Chinese Single-locus Hypertensive subjects with GRK4 A486V are more likely to be non-dippers [166] R65L (rs2960306)

Two-locus
The combination of GRK4 R65L and ACE predicts the hypertensive phenotype [108] American Single-locus Associates with stress-induced reduction in urinary sodium excretion in Black normotensive adolescents [117] Black subject Single-locus Predicts positive blood pressure response to dietary modification in Black subjects with mild-to-moderate hypertension [169] American

Single-locus
Correlates with increased risk for adverse cardiovascular outcomes [164] Swiss Two-locus Patients with GRK4 65L and 142 V need more antihypertensive treatment to reach the same MAP than homozygous carriers of only 1 variant or heterozygous/wild-type carriers of R65L, A142V, and A486V alleles [168] Korean

Single-locus
The GRK4 65 L TT genotype is inversely correlated with hypertension risk [103] Chinese Single-locus Hypertensive subjects with GRK4 R65L are more likely to be non-dippers [166] Other GRK4 renal GRK3 and GRK5 are not different between WKY and SHRs [62].
The role of GRK5 in the regulation of blood pressure is verified in transgenic mice.VSM-specific overexpression of Grk5 increases blood pressure, which is sex-modified because male mice with VSM-Grk5 overexpression have a much greater increase in blood pressure (~26 mm Hg, mean arterial pressure) than female Grk5 mice [122].However, the intracardiac injection of adenovirus that encodes for the RH domain within the amino terminus of Grk5 does not reduce blood pressure levels in SHRs or in WKY rats [123].It should be noted that the role of GRK5 in the regulation of blood pressure is complicated.Mice with global Grk5 ablation have impaired glucose tolerance and insulin sensitivity compared with their WT littermates [124].Therefore, GRK5 may be a positive regulator of insulin resistance, which contributes to the pathogenesis of hypertension [125].In addition, Grk5 overexpression augments the agonist-induced AT 1 R or D 1 R phosphorylation, leading to their desensitization; desensitization of AT 1 R should favor a lower blood pressure while the opposite is true for D 1 R and therefore, these two effects by themselves may not lead to a change in blood pressure [38,41].Cardiac-specific Grk5 expression decreases basal left ventricle systolic pressure, slows left ventricle relaxation, and increases β-AR desensitization, but has no effect on Ang IIinduced contractile response [32].Thus, defining the role of GRK5 in the regulation of blood pressure needs to be studied further.
GRK5 is also associated with other blood pressure regulation-related cardiovascular diseases such as myocardial infarction or cardiometabolic traits such as hyperlipidemia [119,126].Grk5 SNPs are also associated with the race-related therapeutic efficacy of some drugs, including βadrenergic blockers [127,128].

GRK6
GRK6 is ubiquitously expressed in mammalian organs and tissues, including the heart and intestines [71,[129][130][131].There is no direct proof of the role of GRK6 in the regulation of blood pressure.However, there are some reports on the association between GRK6 and hypertension [71].Compared with control rats, total GRK6 expression is increased, and its distribution is reduced in the intercalated disks but increased in the cytoplasm of cardiac myocytes in spontaneously hypertensive heart failure rats [71].This may be involved in abnormal remodeling of cardiac myocytes in hypertensive hypertrophy and failure.The cardiomyocytes of GRK6 but not GRK5 knockout mice have an augmented fractional shortening in response to Ang II [132].
Abnormal proliferation of endothelial cells is part of the vascular remodeling in hypertension that may be related to a decrease in vascular endothelial GRK6 expression caused by miR-27a [133].Cyclic stretching of VSMs, to mimic the pathologically increased stretch in hypertension, causes the transfer of miR-27a via VSMC microparticles to the endothelial cells [133].
As aforementioned, sodium retention, caused by impaired renal sodium excretion, is part of the pathological process in hypertension [134].An increase in intestinal sodium absorption is also part of the increased sodium balance in hypertension, which may be related to increased activity of NHE3 and Na + -K + -ATPase [135,136].Inhibition of GRK6, but not GRK4, prevents the loss of dopamine D 1 -like receptor-mediated inhibition of Cl/HCO3 -exchanger and NHE3 activity in rat intestinal epithelial cells [130,131].In addition, GRK6 regulates insulin homeostasis, an abnormality which participates in the pathogenesis of hypertension [137].GRK6 inhibition increases insulin secretion but reduces insulin processing; Grk6 knockdown reduces cellular insulin levels, and attenuates insulin secretion, but enhances proinsulin secretion consistent with decreased processing [138].However, the proof of the association between GRK6 and hypertension is still weak and needs further exploration.

Modulation of Grks in the regulation of blood pressure
Most studies have focused on the role of GRKs in the regulation of GPCR expression and activity.However, there are studies reporting on how GRKs are regulated.In fact, there are some studies reporting that GRKs per se are modulated by factors, which subsequently regulate blood pressure.For example, long-term exercise in SHRs, beginning at the prehypertensive stage (four weeks old), improves vascular insulin sensitivity via down-regulation of vascular GRK2, which limits the progression of hypertension [58].Activation of the TGF-β signaling cascade in VSMCs increases GRK2 expression, consequently inhibiting Ang II-induced VSMC proliferation and migration [139].We have reported that the expression and activity of GRKs are regulated by several factors, including long-term PM 2.5 exposure, prenatal and paternal PM 2.5 exposure, in utero lipopolysaccharide or cold stress exposure, and maternal diabetes mellitus [49, 87-89, 92, 94].
The expression of GRKs is regulated by transcription factors and signaling molecules.Others and we have reported that renal GRK4 expression is positively regulated by the activation of the transcription factor c-Myc, which binds to the promoter of Grk4 gene [87,90,140].Other transcription factors, such as Foxo1, NF-κB, and cAMP-responsive element binding protein, are also involved in the regulation of transcription of GRKs (GRK2, GRK5, GRK6) [141][142][143].Some key signaling molecules such as protein kinases participate in the modulation of GRK expression.We reported that inhibition of PKC reduces the increased GRK2 expression and normalizes D 1 R function in renal proximal tubule cells from diabetic mother offspring, which may be beneficial in the treatment of maternal diabetes mellitus-programmed hypertension [49].Activation of PKC increases GRK2 protein and activity, causing the dysfunction of endothelium-dependent relaxation to insulin in mouse aorta [144].By contrast, activation of PKC decreases GRK6 promoter activity and expression [143].Posttranscriptional regulation, including ubiquitination, also plays an important role in the regulation of GRKs [145,146].
The regulation of GRK activity or cellular location, independent of its expression, is also important in disease states.Insulin or oxidative stress causes renal D 1 R desensitization and impairs its ability to inhibit Na + -K + -ATPase activity by promoting GRK2 translocation to the plasma membrane [48,147].The increased blood pressure of SHRs is caused, in part, by impaired renal D 1 R function [148], that is due to increased GRK4 activity and sorting nexins (e.g., SNX5 and SNX19) [15,149,150].SNXs play a vital role in receptor trafficking [151].SNX5 directly interacts with renal GRK4 and inhibits its ability to phosphorylate and desensitize D 1 R [149].The nuclear translocation of GRK5 participates in fibroblast activation and cardiac fibrosis; Ang II induces the nuclear translocation of cardiac GRK5 [12].It should be noted that SNPs of GRKs also affect the activity of GRKs.GRK4 variants can increase the activity of GRK4 [28,[77][78][79]93].As aforementioned, increased renal GRK4 activity, caused by GRK4 gene variants, causes hypertension [15,77,78,82,83].The GRK4 variant A486V has been shown to increase the impairment of cardiac function after myocardial infarction [28].A functional GRK2 SNP which inhibits its activity potentiates β-adrenergic receptor-mediated cAMP accumulation and potentially can be used in the treatment of heart failure [152].

GRK inhibition
Because GRKs play a role in the pathogenesis of cardiovascular diseases, including hypertension, many studies have focused on searching selective inhibitors of GRKs, especially GRK2 and GRK5.Some existing drugs have been verified as potential GRK2 inhibitors.For example, paroxetine directly inhibits the activity of GRK2 by binding its active site which stabilizes the kinase domain [153].In hypertension, paroxetine increases β 1 -AR sensitivity and attenuates cardiac hypertrophy by blocking the interaction between GRK2 and β 1 -AR [154].The actions of some GRK inhibitors have been related to their crystal structure.For example, based on the conformation of the HJ loop within the X-ray structure of GRK2, cyclic peptide 7 was found to suppress selectively and potently GRK2 activity [155].The pharmacological inhibition of GRK2 by a cyclic peptide C7 improves cardiac metabolism and function in experimental heart failure [156].Small molecule GRK inhibitors, including paroxetine, Takeda compound 101, and CCG224063, attenuate GRK2-mediated desensitization of vasoconstrictor-induced arterial contractions, highlighting a potential strategy for blood pressure regulation by targeting GRK2 function [157].
Most of the current studies on the inhibition of GRK function have focused on the direct interaction between the inhibitor and the kinase domain of the protein.However, inhibitors of the RH domain of GRK2 have also been reported to reduce the RH-mediated receptor desensitization and GRK2 translocation to the plasma membrane [158,159].These novel inhibitors of phosphorylationindependent actions of GRK2 provide new strategies for the development of innovative pharmacologic therapy for hypertension.
Some studies have also explored the possibility of simultaneous inhibition of two types of GRKs by one inhibitor.For example, analyses of the structures and chemical properties of key residues in the protein crystal structures of GRK2 and GRK5 have determined the specific amino acid distribution within the GRK2/GRK5 target site [160,161].CCG215022, a GRK2 inhibitor, has nanomolar IC50 values against both GRK2 and GRK5.A 2.4 Å crystal structure of the GRK5•CCG215022 complex revealed that the inhibitor binds in the active site similarly to its parent compound [162].The structure and function of GRK4 486V have also been reported; Ser-485 in the kinase C-tail is one of the residues that is responsible for the increased rate of autophosphorylation of GRK4 486V [84].These are promising reports on the generation of selective GRK inhibitors in the treatment of hypertension.

GRKs and pharmacogenomics of hypertension
Over the past decade, pharmacogenetics has been used to guide cardiovascular drug therapy [163].The gene variants of GRKs may be important to guide antihypertensive strategies.The African American Study of Kidney Disease and Hypertension Study showed that in African-American men, but not women, the response to the β 1 -AR blocker metoprolol was more rapid in carriers of GRK4 142V than carriers of GRK4 A142 but the response was decreased in the presence of GRK4 65L [111].The Pharmacogenomic G protein-coupled receptor kinases in hypertension: physiology, pathogenesis, and therapeutic targets Evaluation of Antihypertensive Responses trial showed that the response of hypertensive patients to β-blockers is influenced by GRK4 variants.In African-Americans and Caucasians, increasing number of copies of the GRK4 variant 65L-142V haplotype is associated with reduced response to β-blocker monotherapy with atenolol [164].Moreover, all three GRK4 variants (65L, 142V, and 486V) are associated with increased risk for the primary outcome of first occurrence of all cause death, nonfatal myocardial infarction, or nonfatal stroke in American whites and Hispanics [164].Another study in hypertensive Japanese showed that patients with hGRK4 142V have a greater decrease in SBP in response to angiotensin receptor blockers (ARBs) than non-carrier subjects.However, those with GRK4 486V are less likely to achieve the blood pressure goal in response to ARBs than those without GRK4 variants [165].Our study also found that non-dipper circadian rhythm is positively associated with the presence of GRK4 variants.Moreover, patients with these three GRK4 variants have a better antihypertensive response to candesartan, an ARB, than those with GRK4 WT gene [166].
Regarding the antihypertensive effect of diuretics, one study showed that subjects with more than three GRK4 gene variants (A142V, A486V, and R65L) have a greater increase in potassium and sodium excretion in response to hydrochlorothiazide than those with three or less than three GRK4 gene variants [167].In another study, hypertensive subjects with GRK4 65L and 142V needed more antihypertensive therapy, especially diuretics, to reach the same mean arterial pressure as did the heterozygous and wild-type carriers of GRK4 [168].Other GRK variants are reported to be associated with therapeutic effect of antihypertensive drugs.African-American carriers of GRK2 rs4930416 have a greater decrease in both SBP and DBP in response to hydrochlorothiazide while European-Americans with GRK2 rs1894111 have a greater decrease in both SBP and DBP in response to hydrochlorothiazide [16].African-American or European-American carriers of GRK5Gln41, relative to homozygous carriers of GRK5Leu41, do not respond differently to hydrochlorothiazide or atenolol but those with GRK5Leu41 have a 46.5% risk reduction for adverse cardiovascular outcomes compared with GRK5 Gln41 homozygotes, independent of treatment [16].
GRK4 variants are also associated with the blood pressure response to lifestyle modification.For example, Rayner et al. found that in South African Blacks 50-75 years old with mild-to-moderate hypertension, GRK4 polymorphisms, A142V and R65L, can predict blood pressure response to dietary reduction of dietary salt intake.Hypertensive subjects with wild-type GRK4 A142 and GRK4 R65 or heterozygous GRK4 A142V and GRK4 R65L but no homozygous GRK4 142V and GRK4 65L have a reduction in their blood pressures in response to a low salt diet [169].Increases ear inflammation [177] α-AR α-adrenergic receptor, AdGRK5-NT an adenovirus that encodes for the RH domain within the amino terminal of GRK5, adipoR1 adiponectin receptor 1,

Limitations of Grks research
There are several limitations in the current GRK research.First, there are only a few reports using organ-specific knockout or overexpression GRK mice, which are better to study the role of GRKs in the regulation of blood pressure.Second, the mechanism by which different GRKs polymorphisms affect enzymatic activity remains still unknown, which may be related with the crystal structure of GRKs polymorphisms.Especially, the reason why different GRK4 variants exhibit different salt-sensitive phenotypes in blood pressure regulation needs to be studied in the future.Third, GRK2-mediated effects in the regulation of blood pressure is complicated, and its role in hypertension needs to be clarified more clearly.Fourth, the regulatory mechanisms of GRK remain unclear.

Conclusions
In summary, increasing pieces of evidence demonstrate that GRKs, by regulating different substrates, play important roles in the regulation of blood pressure.Various genetically modified animal models confirm the key role of GRKs in the regulation of blood pressure (Table 3).Aberrant expression and/or activity of GRKs in the cardiovascular system and kidney participate in the pathogenesis of hypertension.Genetic association studies have found associations between GRK gene variants and hypertension.Moreover, individual GRK variants also affect the response to antihypertensive treatment.Increased understanding of the role of GRKs in the regulation of blood pressure may provide novel insights into the pathogenesis of hypertension and new strategies for the prevention and treatment of hypertension.

Table 1
GRK substrates related to the regulation of blood pressure

Table 2
GRK4 variants in hypertensive and normotensive subjects

Table 3
Effects of GRK modification on blood pressure and related substrates